Department of Translational Medicine and Department of Biochemistry and Molecular Biology, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505; Stem Cell Immunomodulation Research Center, University of Ulsan College of Medicine, Seoul 05505, Korea.
Department of Cell and Genetic Engineering, Asan Medical Institute of Convergence Science and Technology, Asan Medical Center, University of Ulsan College of Medicine, Seoul 05505; Center for Genomic Integrity, Institute for Basic Science, Ulsan 44919; Stem Cell Immunomodulation Research Center, University of Ulsan College of Medicine, Seoul 05505, Korea.
BMB Rep. 2024 Jan;57(1):60-65. doi: 10.5483/BMBRep.2023-0113.
The CRISPR-Cas9 system has significantly advanced regenerative medicine research by enabling genome editing in stem cells. Due to their desirable properties, mesenchymal stem cells (MSCs) have recently emerged as highly promising therapeutic agents, which properties include differentiation ability and cytokine production. While CRISPR-Cas9 technology is applied to develop MSC-based therapeutics, MSCs exhibit inefficient genome editing, and susceptibility to plasmid DNA. In this study, we compared and optimized plasmid DNA and RNP approaches for efficient genome engineering in MSCs. The RNP-mediated approach enabled genome editing with high indel frequency and low cytotoxicity in MSCs. By utilizing Cas9 RNPs, we successfully generated B2M-knockout MSCs, which reduced T-cell differentiation, and improved MSC survival. Furthermore, this approach enhanced the immunomodulatory effect of IFN-r priming. These findings indicate that the RNP-mediated engineering of MSC genomes can achieve high efficiency, and engineered MSCs offer potential as a promising therapeutic strategy. [BMB Reports 2024; 57(1): 60-65].
CRISPR-Cas9 系统通过对干细胞进行基因组编辑，极大地推动了再生医学研究。间充质干细胞（MSCs）由于其理想的特性，最近成为极具前途的治疗剂，其特性包括分化能力和细胞因子产生能力。虽然 CRISPR-Cas9 技术被应用于开发基于 MSC 的治疗方法，但 MSC 表现出基因组编辑效率低下和对质粒 DNA 的易感性。在这项研究中，我们比较和优化了质粒 DNA 和 RNP 方法，以实现 MSC 中的高效基因组工程。RNP 介导的方法可在 MSC 中实现高插入缺失频率和低细胞毒性的基因组编辑。通过使用 Cas9 RNPs，我们成功地生成了 B2M 敲除 MSC，这降低了 T 细胞分化，并提高了 MSC 的存活率。此外，该方法增强了 IFN-r 引发的免疫调节作用。这些发现表明，MSC 基因组的 RNP 介导工程可以实现高效率，并且工程 MSC 有望成为一种有前途的治疗策略。[BMB 报告 2024；57(1):60-65]。
